Glucokinase (GK) is one of four hexokinases found in mammals [Colowick, S. P., in The Enzymes, Vol. 9 (P. Boyer, ed.) Academic Press, New York, N.Y., pages 1-48, 1973]. The hexokinases catalyze the first step in the metabolism of glucose, i.e., the conversion of glucose to glucose-6-phosphate. Glucokinase has a limited cellular distribution, being found principally in pancreatic xcex2-cells and liver parenchymal cells. In addition, GK is a rate-controlling enzyme for glucose metabolism in these two cell types that are known to play critical roles in whole-body glucose homeostasis [Chipkin, S. R., Kelly, K. L., and Ruderman, N. B. in Joslin""s Diabetes (C. R. Khan and G. C. Wier, eds.), Lea and Febiger, Philadelphia, Pa., pages 97-115, 1994]. The concentration of glucose at which GK demonstrates half-maximal activity is approximately 8 mM. The other three hexokinases are saturated with glucose at much lower concentrations ( less than 1 mM). Therefore, the flux of glucose through the GK pathway rises as the concentration of glucose in the blood increases from fasting (5 mM) to postprandial (≈10-15 mM) levels following a carbohydrate-containing meal [Printz, R. G., Magnuson, M. A., and Granner, D. K. in Ann. Rev. Nutrition Vol. 13 (R. E. Olson, D. M. Bier, and D. B. McCormick, eds.), Annual Review, Inc., Palo Alto, Calif., pages 463-496, 1993]. These findings contributed over a decade ago to the hypothesis that GK functions as a glucose sensor in xcex2-cells and hepatocytes (Meglasson, M. D. and Matschinsky, F. M. Amer. J. Physiol. 246, E1-E13, 1984). In recent years, studies in transgenic animals have confirmed that GK does indeed play a critical role in whole-body glucose homeostasis. Animals that do not express GK die within days of birth with severe diabetes while animals overexpressing GK have improved glucose tolerance (Grupe, A., Hultgren, B., Ryan, A. et al., Cell 83, 69-78, 1995; Ferrie, T., Riu, E., Bosch, F. et al., FASEB J., 10, 1213-1218, 1996). An increase in glucose exposure is coupled through GK in xcex2-cells to increased insulin secretion and in hepatocytes to increased glycogen deposition and perhaps decreased glucose production.
The finding that type II maturity-onset diabetes of the young (MODY-2) is caused by loss of function mutations in the GK gene suggests that GK also functions as a glucose sensor in humans (Liang, Y., Kesavan, P., Wang, L. et al., Biochem. J. 309, 167-173, 1995). Additional evidence supporting an important role for GK in the regulation of glucose metabolism in humans was provided by the identification of patients that express a mutant form of GK with increased enzymatic activity. These patients exhibit a fasting hypoglycemia associated with an inappropriately elevated level of plasma insulin (Glaser, B., Kesavan, P., Heyman, M. et al., New England J. Med. 338, 226-230, 1998). While mutations of the GK gene are not found in the majority of patients with type II diabetes, compounds that activate GK and, thereby, increase the sensitivity of the GK sensor system will still be useful in the treatment of the hyperglycemia characteristic of all type II diabetes. Glucokinase activators will increase the flux of glucose metabolism in xcex2-cells and hepatocytes, which will be coupled to increased insulin secretion. Such agents would be useful for treating type II diabetes.
This invention provides a compound, comprising a substituted hydantoin of the formula: 
wherein
R1 is a five- or six-membered aromatic heterocyclic ring having one to three heteroatoms selected from nitrogen, oxygen, and sulfur, which ring is unsubstituted or substituted with halo, amino, hydroxylamino, nitro, cyano, sulfonamido, lower alkyl, perfluoro lower alkyl, lower alkyl thio, perfluoro-lower alkyl thio, lower alkyl sulfonyl, perfluoro-lower alkyl sulfonyl, lower alkyl sulfinyl, or xe2x80x94(R5)nxe2x80x94C(O)xe2x80x94OR6;
R2 is a cycloalkyl ring containing from 5 to 7 carbon atoms;
R3 is hydrogen, lower alkyl, a cycloalkyl ring containing from 5 to 7 carbon atoms, unsubstituted aryl, aryl substituted with halo or hydroxy, or an unsubstituted five- or six-membered aromatic heterocyclic ring having one or two heteroatoms selected from nitrogen, oxygen, and sulfur;
R4 is hydrogen, lower alkyl, or R3 and R4 together with the carbon atom to which they are attached form a cycloalkyl ring containing 5 to 7 carbon atoms;
R5 is xe2x80x94C(O)xe2x80x94 or lower alkyl;
R6 is lower alkyl;
n is 0 or 1; * and ** each designate an asymmetric centers,
and pharmaceutically acceptable salts thereof.
The compounds of Formula I have been found to activate glucokinase. Glucokinase activators are useful for increasing insulin secretion in the treatment of type II diabetes. Therefore compounds of this invention are useful to increase insulin secretion in view of their activity as glucokinase activators.
This invention is directed to compounds of Formula I above. The invention is particularly directed to compounds as follows, where:
R2 and R3 are both cyclohexyl, or
R3, when it is lower alkyl, is methyl, ethyl, propyl, or butyl, or
R4, when it is lower alkyl, is methyl or ethyl (especially compounds where R3 and R4 are both so defined), or
R1, when substituted, is substituted with halo, lower alkyl, or xe2x80x94(R5)nxe2x80x94C(O)xe2x80x94OR6, or compounds where any two or more, or all, of these conditions are met. For any compound of this invention where R1, R2, or R3 are not specified, it is preferred that the variable is as described in this paragraph.
Certain preferred compounds of Formula I include a compound where R1 is substituted or unsubstituted thiazolyl (Compound A). Among the embodiments of Compound A are those compounds where R1 is thiazolyl substituted with halo, lower alkyl, or xe2x80x94(R5)nxe2x80x94C(O)xe2x80x94OR6, and especially with xe2x80x94(R5)nxe2x80x94C(O)xe2x80x94OR6. (Compound A-1). In Compound A-1, it is preferred that R2 is cyclopentyl or cyclohexyl. It is also preferred that R3 is cyclopentyl or cyclohexyl. It is preferred that R4 is hydrogen. It is especially preferred that R2 and R3 are cyclohexyl.
In preferred embodiments of Compound A-1, R2 and R3 are cyclopentyl or cyclohexyl, and R4 is hydrogen (Compound A-1a). In one embodiment of Compound A-1 a, n is 0 (e.g., the thiazolyl is substituted with xe2x80x94C(O)xe2x80x94OR6). Examples of such compounds are
(S,S)-2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester.
(S,S)-2-[[2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl)-3-cyclopentylpropanoyl]amino]thiazole-4-carboxylic acid methyl ester.
(S,S)-2-[[3-cyclopentyl-2-[4-(cyclopentyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester.
(S,S)-2-[[3-cyclohexyl-2-[4-(cyclopentyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester.
In such a compound, R2 and R3 may both be cyclohexyl, for example (S,S)-2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino] thiazole-4-carboxylic acid methyl ester.
In another embodiment of Compound A-1a, R5 is xe2x80x94C(O)xe2x80x94 or lower alkyl (e.g., the thiazolyl is substituted with xe2x80x94C(O)xe2x80x94C(O)xe2x80x94OR6 or -lower alkyl-C(O)xe2x80x94OR6). In addition, in such compounds R2 and R3 may be cyclohexyl. Examples of such compounds are
(S,S)-[2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-yl]oxoacetic acid ethyl ester and
(S,S)-[2-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]thiazole-4-yl]acetic acid ethyl ester.
In another embodiment of Compound A1, R2 is cyclopentyl or cyclohexyl (Compound A-1b). In one embodiment of Compound A-1b, R3 is substituted or unsubstituted phenyl and R4 is hydrogen. Examples of these compounds are
(S,S)-2-[[2-(4-benzyl-2,5-dioxoimidazolidin-1-yl)-3-cyclohexylpropanoyl]amino]thiazole-4-carboxylic acid methyl ester.
2-[[(S)-2-[(R)-4-(4-chlorobenzyl)-2,5-dioxoimidazolidin-1-yl]-3-cyclohexylpropanoyl]amino]thiazole-4-carboxylic acid methyl ester.
(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(4-hydroxybenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester.
(S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(3-hydroxybenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester, and
2-[[(S)-3-cyclohexyl-2-[(R,S)-2,5-dioxo-4-(4-fluorobenzyl)imidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester.
In another embodiment of Compound A-1b, at least one of R3 and R4 are lower alkyl. Examples of such compounds are
(S)-2-[[3-cyclohexyl-2-(4,4-dimethyl-2,5-dioxoimidazolidin-1-yl)propanoyl]amino]thiazole-4-carboxylic acid methyl ester, and
2-[[(S)-3-cyclohexyl-2-[(R)-2,5-dioxo-4-propylimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester.
In yet another embodiment of Compound A-1b, R3 is naphthyl and R4 is hydrogen. An example of such a compound is (S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(naphthalen-2-yl)methylimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester.
In another embodiment of Compound A-1b, R3 and R4 together with the carbon atoms to which they are attached form a cycloalkyl ring containing 5 to 7 carbon atoms. An example of such a compound is (S)-2-[[3-cyclohexyl-2-(2,4-dioxo-1,3-diazaspiro[4.4]non-3-yl)propanoyl]amino]thiazole-4-carboxylic acid methyl ester.
And in another embodiment of Compound A-1b, R3 is an unsubstituted five- or six-membered aromatic heterocyclic ring having one or two heteroatoms selected from nitrogen, oxygen, and sulfur. An example of such a compound is (S,S)-2-[[3-cyclohexyl-2-[2,5-dioxo-4-(thiophen-2-yl)methylimidazolidin-1-yl]propanoyl]amino]thiazole-4-carboxylic acid methyl ester.
In one embodiment of Compound A (a compound of Formula I wherein R1 is substituted or unsubstituted thiazolyl), R1 is unsubstituted thiazolyl (Compound A-2). It is preferred that R2 and R3 are cyclohexyl and R4 is hydrogen. An example of such a Compound A-2 is (S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(thiazole-2-yl)propanamide.
In other preferred compounds of Formula I, R1 is substituted or unsubstituted pyridine (Compound B). It is preferred that R2 is cyclopentyl or cyclohexyl, especially cyclohexyl. It is also preferred that R3 is cyclopentyl or cyclohexyl, especially cyclohexyl. It is preferred that R4 is hydrogen.
In one embodiment of Compound B, R2 is cyclohexyl. In such a compound where R2 is cyclohexyl, it is preferred that R3 is cyclohexyl and R4 is hydrogen (Compound B-1).
In one embodiment of Compound B-1, R1 is substituted pyridine. Preferably the pyridine is substituted with xe2x80x94(R5)nxe2x80x94C(O)xe2x80x94OR6, especially where n is 0 and R6 is lower alkyl, such as methyl (e.g., methoxycarbonyl). Examples of such compounds are
(S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(5-methylpyridin-2-yl)propanamide.
(S,S)-6-[[3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanoyl]amino]nicotinic acid methyl ester, and
(S,S)-N-(5-chloropyridin-2-yl)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]propanamide.
In another embodiment of Compound B-1, R1 is unsubstituted pyridine. An example of such a compound is (S,S)-3-cyclohexyl-2-[4-(cyclohexyl)methyl-2,5-dioxoimidazolidin-1-yl]-N-(pyridin-2-yl)propanamide.
In the compound of Formula I the xe2x80x9c*xe2x80x9d and xe2x80x9c**xe2x80x9d illustrate the two separate asymmetric centers. The (S) enantiomer at the position designated by xe2x80x9c**xe2x80x9d is preferred. However the compounds of this invention may be pure (R)(R), pure (S)(S), pure (R)(S), pure (S)(R) or any mixture of pure enantiomers.
As used throughout this application unless otherwise specified, the term xe2x80x9clower alkylxe2x80x9d includes both straight chain and branched chain alkyl groups having from 1 to 6 or 1 to 7 carbon atoms, such as methyl, ethyl, propyl, isopropyl, preferably methyl and ethyl. Unless otherwise specified, propyl is taken to include both forms of propyl (e.g., isopropyl, n-propyl) and butyl is taken to include all forms of butyl (e.g., isobutyl, n-butyl, tert-butyl). Preferred at R3 is methyl, ethyl, propyl, or butyl. Preferred at R4 is methyl or ethyl.
The term xe2x80x9ccycloalkyl ringxe2x80x9d may be a ring of from three to seven carbon atoms, but preferably from five to seven carbon atoms, especially cyclopentyl, cyclohexyl, cyclobutyl and cyclopropyl. The more preferable cycloalkyl groups contain from 5 to 6 carbon atoms, e.g., cyclopentyl and cyclohexyl, and cyclohexyl is most preferable. As used herein, xe2x80x9cperfluoro-lower alkylxe2x80x9d means any lower alkyl group wherein all of the hydrogens of the lower alkyl group are substituted or replaced by fluoro. Among the preferred perfluoro-lower alkyl groups are trifluoromethyl, pentafluoroethyl, heptafluoropropyl, etc.
As used herein, xe2x80x9clower alkyl thioxe2x80x9d means a lower alkyl group as defined above where a thio group is bound to the rest of the molecule. Similarly xe2x80x9cperfluoro-lower alkylxe2x80x9d thio means a perfluoro-lower alkyl group as defined above where a thio group is bound to the rest of the molecule. As used herein, xe2x80x9clower alkyl sulfonylxe2x80x9d or xe2x80x9clower alkyl sulfinylxe2x80x9d means a lower alkyl group as defined above where a sulfonyl or sulfinyl group is bound to the rest of the molecule. Similarly xe2x80x9cperfluoro-lower alkyl sulfonylxe2x80x9d means a perfluoro-lower alkyl group as defined above where a sulfonyl group is bound to the rest of the molecule.
When R3 and R4 together with the carbon atom to which they are attached form a cycloalkyl ring containing five to seven carbon atoms, this includes the ring carbon atom and the methylene linking the ring carbon atom and R4 such that if R3 and R4 are each methylene, cyclobutyl is formed. If R3 is methylene and R4 is ethylene, cyclopentyl is formed, etc.
As used herein, the terms xe2x80x9chalogenxe2x80x9d or xe2x80x9chaloxe2x80x9d unless otherwise specified, designates all four halogens, i.e. fluorine, chlorine, bromine and iodine.
R1 is, and R3 can be any five- or six-membered aromatic heterocyclic ring containing from one to three, preferably from one to two, heteroatoms selected from the group consisting of sulfur, oxygen or nitrogen. Any such five- or six-membered aromatic heterocyclic ring can be used in accordance with this invention. Among the preferred rings for R1 are thiazole and pyridine (especially pyridine), and a preferred ring for R3 is thiophene. R1, and R3 when R3 is a heterocyclic ring, is connected to the remainder of the molecule of Formula I through a ring carbon atom. When R1 is substituted as described in Formula I, the substituent is on a ring carbon atom. R1 is preferably monosubstituted, but may be di or tri substituted. A preferred substituent, especially for pyridine, is lower alkoxy (preferably methoxy) carbonyl.
As used herein the term xe2x80x9carylxe2x80x9d signifies an aromatic hydrocarbon ring having six or ten carbon atoms such as phenyl or naphthyl.
The compounds of this invention may be produced by the reaction schemes provided below.
The term xe2x80x9cresinxe2x80x9d designates any conventional polymer resin which has suitable characteristics for use in solid phase peptide synthesis. A resin with the suitable characteristics is inert, physically stable, insoluble in inorganic solvents, and has a linker functionality which is labile under known chemical conditions. Preferred are polystyrene resins having chemically labile functional linkers such as trityl resins and especially Wang resins.
The term xe2x80x9camino protecting groupxe2x80x9d designates any conventional amino protecting group which can be cleaved to yield the free amino group. The preferred protecting groups are the conventional amino protecting groups utilized in peptide synthesis. Especially preferred are those amino protecting groups which are cleavable under treatment with secondary dialkyl amines. A particularly preferred amino protecting group is 9H-fluoren-9-ylmethoxy carbamate.
xe2x80x9cOrthogonalxe2x80x9d is the term used to describe the relationship of the amino protecting group to the resin. The resin and the amino protecting group must be compatible, in that the resin-peptide bond and the amino protecting group should not labile under the same conditions. During synthesis of a given compound, one should be able to cleave the amino protecting groups off the compound while leaving the compound attached to the resin. In other words, the conditions under which the amino protecting group comes off the compound should not also cause the compound to come off the resin. It is preferred that the amino protecting group be cleavable under basic or weakly acidic conditions, because the preferred Wang-type resins are cleavable under strongly acidic conditions (i.e. about pH 0 to about pH 1) A skilled person will readily be able to determine the necessary conditions to select an orthogonal amino protecting group-resin set.
The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d as used herein include any salt with both inorganic or organic pharmaceutically acceptable acids such as hydrochloric acid, hydrobromic acid, nitric acid, sulfuric acid, phosphoric acid, citric acid, formic acid, maleic acid, acetic acid, succinic acid, tartaric acid, methanesulfonic acid, para-toluene sulfonic acid and the like. The term xe2x80x9cpharmaceutically acceptable saltsxe2x80x9d also includes any pharmaceutically acceptable base salt such as amine salts, trialkyl amine salts and the like. Such salts can be formed quite readily by those skilled in the art using standard techniques.
In accordance with this invention, the compounds of Formula I are produced by the following reaction schemes. Any compound of Formula I may be produced as shown in Reaction Scheme 1. The compounds of Formula I-A are produced as shown in Reaction Scheme 2. Reaction Scheme 3 shows how to produce N-Fmoc-aminothiazole-4-carboxylic acid, which is compound 3 of Scheme 2 where PG is the protecting group Fmoc. 
wherein R1, R2, R3 and R4 are as previously described and PG1 and PG2 are amine protecting groups which may or may not be equivalent, that are removable under conditions compatible with the Linker-O bond. 
wherein R2, R3, R4 and R6 are as previously described and PG, PG1 and PG2 are amine protecting groups which may or may not be equivalent, that are removable under conditions compatible with the linker-O bond and where the ring A represents a five or six membered heteroaromatic ring having one, two or three hetero atoms selected from nitrogen, oxygen or sulfur. 
The synthesis of the compounds of this invention may be carried out by a procedure whereby each amino acid in the desired sequence is added one at a time in succession to another amino acid or residue thereof or by a procedure whereby peptide fragments with the desired amino acid sequence are first synthesized conventionally and then condensed to provide the compound.
Such conventional procedures for synthesizing the novel compounds of the present invention include for example any solid phase peptide synthesis method. In such a method the synthesis of the novel compounds can be carried out by sequentially incorporating the desired amino acid residues one at a time into the growing peptide chain according to the general principles of solid phase methods [Merrifield, R. B., J. Amer. Chem. Soc. 1963, 85, 2149-2154; Barany et al., The Peptides, Analysis, Synthesis and Biology, Vol. 2, Gross, E. and Meienhofer, J., Eds. Academic Press 1-284 (1980); Bunin, B., Combinatorial Index, Academic Press (1998)].
Common to chemical syntheses of peptides is the protection of reactive side chain groups of the various amino acid moieties with suitable protecting groups, which will prevent a chemical reaction from occurring at that site until the protecting group is ultimately removed. Usually also common is the protection of the alpha amino group of an amino acid or fragment while that entity reacts at the carboxyl group, followed by the selective removal of the alpha amino protecting group and allow a subsequent reaction to take place at that site. While specific protecting groups are mentioned below in regard to the solid phase synthesis method, it should be noted that each amino acid can be protected by any protective group conventionally used for the respective amino acid in solution phase synthesis.
For example, alpha amino groups may be protected by a suitable protecting group selected from aromatic urethane-type protecting groups, such as benzyloxycarbonyl (Z) and substituted benzyloxycarbonyl, such as p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, p-biphenyl-isopropoxycarbonyl, 9-fluorenylmethoxycarbonyl (Fmoc) and p-methoxybenzyloxycarbonyl (Moz); aliphatic urethane-type protecting groups, such as t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl, isopropoxycarbonyl, and allyloxycarbonyl. In the present case, Fmoc is the most preferred for alpha amino protection. Guanidino groups may be protected by a suitable protecting group selected from nitro, p-toluenesulfonyl (Tos), Z, pentamethylchromanesulfonyl (Pmc), adamantyloxycarbonyl, and Boc. Pmc is the most preferred for arginine (Arg).
The solvents dichloromethane, dimethylformamide (DMF) and N-methylpyrrolidinone and toluene may be purchased from Fisher or Burdick and Jackson and may be used without additional distillation. Trifluoroacetic acid was purchased from Halocarbon or Fluka and used without further purification. Diisopropylcarbodiimide and diisopropylethylamine (DIPEA) was purchased from Fluka or Aldrich and used without further purification. 1-Hydroxybenzotriazole (HOBT) may be purchased from Sigma Chemical Co. and used without further purification. Protected amino acids, unless otherwise specified, are generally preferably of the L configuration and may be obtained commercially from Bachem, Advanced ChemTech, or Neosystem. Such amino acids may also be chemically synthesized using any one of several well known methods of amino acid synthesis. The configuration of the amino acids 5 and 7 used to prepare a given compound of this invention will determine the configuration of the ** and * positions respectively of Formula I. Therefore, it is useful to select the amino acid configuration with the desired final configuration in mind. L amino acids have the (S) absolute configuration and D amino acids have the (R) absolute configuration.
Compounds of this invention may be prepared using solid phase synthesis following the principles and general methods described by Merrifield or by Bunin, although other equivalent chemical synthesis known in the art could be used as previously mentioned. Solid phase synthesis is commenced from the C-terminal end of the peptide by coupling a N-protected amino acid to a suitable resin. Such a starting material can be prepared by attaching an N-protected amino acid by an ester linkage to a p-benzyloxybenzyl alcohol (Wang) resin, or by an amide bond between an Fmoc-Linker, such as p-[(R,S)-xcex1-[1-(9H-fluoren-9-yl)-methoxyformamido]-2,4-dimethyloxybenzyl]-phenoxyacetic acid (Rink linker) to a benzhydrylamine (BHA) resin. Preparation of the hydroxymethyl resin is well known in the art. Wang resin supports are commercially available and generally used when the desired peptide being synthesized has an ester or a substituted amide at the C-terminus. To form the starting resin bound amino acid, a Fmoc N-protected amino acid is activated by the formation of a mixed anhydride which in turn couples with the hydroxymethyl resin though an ester bond. Several reagents are used to form mixed anhydrides in which the carbonyl group originating from the C-terminal amino acid is preferentially activated to nucleophilic attack by the hydroxymethyl residues in the Wang resin, through either electronic or steric effects. For example, appropriate compounds used in the formation of the mixed anhydrides are trimethylacetyl chloride, 2,6-dichlorobenzoyl chloride and 2,4,6-trichlorobenzoyl chloride, preferably 2,6-dichlorobenzoyl chloride.
Subsequently, the amino acids or mimetics are then coupled onto the Wang resin using the Fmoc protected form of the amino acid or mimetic, with 2-5 equivalents of amino acid and a suitable coupling reagent. After each coupling, the resin may be washed and dried under vacuum. Loading of the amino acid onto the resin may be determined by amino acid analysis of an aliquot of Fmoc-amino acid resin or by determination of Fmoc groups by UV analysis.
The resins are carried through one or two cycles to add amino acids sequentially. In each cycle, the N-terminal Fmoc protecting group is removed under basic conditions from the resin bound amino acid. A secondary amine base such as piperidine, piperazine or morpholine, preferably piperidine (20-40% v/v) in an inert solvent, for example, N,N-dimethylformamide is particularly useful for this purpose. Following the removal of the alpha amino protecting group, the subsequent protected amino acids are coupled stepwise in the desired order to obtain an N-Fmoc protected peptide-resin. The activating reagents used for coupling of the amino acids in the solid phase synthesis of the peptides are well known in the art. For example, appropriate coupling reagents for such syntheses are [(benzotriazol-1-yl)oxy]tris(dimethylamino) phosphonium hexafluorophosphate (BOP), [(benzotriazol-1-yl)oxy]tis(pyrrolidino)-phosphonium hexafluorophosphate (PyBOP), O-(1H-benzotriazole-1-yl)-N,N,Nxe2x80x2,Nxe2x80x2-tetramethyluronium hexafluorophosphate (HBTU), and diisopropylcarbodiimide (DIC), preferably HBTU and DIC. Other activating agents as described by Barany and Merrifield [The Peptides, Vol. 2, J. Meienhofer, ed., Academic Press, 1979, pp 1-284] may be utilized. The couplings are conveniently carried out in an inert solvent, such as N,N-dimethylformamide or N-methylpyrrolidinone, preferably N-methylpyrrolidinone, optionally in the presence of a substance that minimizes racemization and increases the rate of reaction. Among such substances are 1-hydroxybenzotriazole (HOBT), 3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HOOBT), 1-hydroxy-7-azabenzotriazole (HOAT), and N-hydroxysuccinimide (HOSu). In the present instance, HOBT is preferred.
The protocol for a typical coupling cycle is as follows (Method B):
Solvents for all washings and couplings may be measured to volumes of, for example, 10-20 ml/g resins. Coupling reactions throughout the synthesis may be monitored by assays, such as the Kaiser ninhydrin test, to determine extent of completion [Kaiser et at. Anal. Biochem. 1970, 34, 595-598].
When the requisite number of amino acid units have been assembled on the resin, the N-terminal Fmoc group may be cleaved using Steps 1-4 of Method B and the deprotected amine is reacted with phosgene or a phosgene equivalent to form an isocyanate. The reagent of choice in this transformation is trichloromethyl chloroformate (diphosgene). The reaction is carried out in an inert solvent, for example dichloromethane, in the presence of a proton acceptor. When a suspension of the resin bound isocyanate is heated, cyclization occurs wherein the isocyanate moiety condenses with the nitrogen of the neighboring amide group to form a 2,5-dioxoimidazolidine ring.
The compounds may be cleaved from the resin by the following procedure, conditions which also remove other protecting groups if they are present. The peptide-resins are shaken in a mixture (1:1) of trifluoroacetic acid in dichloromethane, optionally in the presence of a cation scavanger, for example ethanedithiol, dimethylsulfide, anisole or triethylsilane, at room temperature for 60 min. The cleavage solution may be filtered free from the resin, concentrated to dryness, and the product then used per se in subsequent transformations as shown in Reaction Scheme 1 and Reaction Scheme 2.
Compounds of Formula 1 can be prepared by the methods outlined in Reaction Scheme 1 and Reaction Scheme 2. Reaction Scheme 2 is a general procedure that can be used to prepare all compounds embodied by Formula 1, but in the present case, it is particularly useful in the preparation of compounds where R1 is varied while R2 and R3 are limited to cycloalkyl and R4 is hydrogen. Reaction Scheme 1 is used in the preparation of compounds of Formula I-A.
In Reaction Scheme 2, an N-protected-amino acid 3 (see Reaction Scheme 3) is converted to a mixed anhydride on treatment with 2,6-dichlorobenzoyl chloride in the presence of Wang resin 2 and a proton acceptor, such as triethylamine, diisopropylethylamine or pyridine, preferably pyridine to give the resin bound amino acid of structure 4. The reaction is conveniently carried out in an inert solvent for example N,N-dimethylformamide or N-methylpyrrolidinone, preferably N-methylpyrrolidinone at from zero degrees to room temperature, most conveniently at room temperature. The conversion of 4 to the resin bound compound of structure 6 can be achieved by using the protocol outlined in method B. Thus after N-deprotection of the resin bound amino acid of structure 4 with piperidine in N,N-dimethylformamide, the product is then acylated with the Nxcex1-protected amino acid of structure 5 in the presence of diisopropylcarbodiimide and HOBT in N-methylpyrrolidinone. The deprotection and N-acylation is carried out at a temperature between about zero degrees and about room temperature, preferably at about room temperature. By using the coupling cycle described above for the conversion of 4 to 6 the Nxcex1-protected amino acids of structure 7 is incorporated into the resin bound compound of structure 6. Thus compounds of structure 6 are sequentially deprotected with piperidine in N,N-dimethylformamide and then coupled with compounds of structure 7 in the presence of diisopropylcarbodiimide and 1-hydroxybenzotriazole in N-methylpyrrolidinone at a temperature between about zero degrees and about room temperature, preferably at about room temperature to afford the resin bound compounds of structure 8.
The N-terminus protecting group PG2 in the compounds of structure 8 was removed on treatment with a secondary amine base, preferably piperidine in an inert solvent (preferably N,N-dimethylformamide) and then was reacted with phosgene or a phosgene equivalent reagent, to ultimately yield in a two step sequence, the 2,5-dioxoimidazolidines of structure 10. The reaction to give the intermediate isocyanate 9 is conveniently carried out using trichloromethyl chloroformate (diphosgene) in an inert solvent, for example, a halogenated hydrocarbon in the presence of a proton acceptor, for example, pyridine, triethylamine or diisopropylethylamine, preferably diisopropylethylamine at a temperature between about zero degrees and about room temperature, preferably at about room temperature. The thermally induced cyclization of the intermediate isocyanates is performed by heating a suspension of the resin bound isocyanates of structure 9 in an inert solvent, for example toluene, at a temperature of from between 50xc2x0 C. and the reflux temperature of the mixture, preferably at about 70xc2x0 C. to give the resin bound compounds of structure 10.
Cleavage of the assembled peptidic residue 10 from the solid support to give the acids of structure 11 is achieved by shaking a suspension of 10 in a strong acid, for example methanesulfonic acid, hydrofluoric acid or trifluoroacetic acid, preferably trifluoroacetic acid optionally in the presence of a cation scavenger and an inert co-solvent, for example dichloromethane. The reaction is conveniently run at a temperature between about zero degrees and about room temperature, preferably at about room temperature.
To complete the synthesis, the acid of structure 11 is reacted with an alcohol (R6OH) to form the ester 1. The esterification can be accomplished using many of the methods well known to those of average skill in the field of organic chemistry. The conversion is conveniently carried out using a coupling reagent, for example one of the many useful carbodiimides, preferably the water soluble 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, optionally using R6OH or a mixture of R6OH and a inert co-solvent, e.g., dichloromethane, as the reaction medium. The reaction is run at a temperature between about zero degrees and about room temperature, preferably at about room temperature.
In a similar fashion, Reaction Scheme 1, the Nxcex1-protected amino acids of structure 5  is converted to a mixed anhydride on treatment with 2,6-dichlorobenzoyl chloride in the presence of Wang resin 2 and a proton acceptor, such as triethylamine, diisopropylethylamine or pyridine, preferably pyridine to give the resin bound amino acid of structure 12. The reaction is conveniently carried out in an inert solvent for example N,N-dimethylformamide or N-methylpyrrolidinone, preferably N-methylpyrrolidinone at from zero degrees to room temperature, most conveniently at room temperature. The conversion of 12 to the resin bound compound of structure 13 can be achieved by using the protocol outlined in method B. Thus after N-deprotection of the resin bound amino acid of structure 12 with piperidine in N,N-dimethylformamide, the product is then acylated with the N-protected amino acid of structure 7 in the presence of diisopropylcarbodiimide and HOBT in N-methylpyrrolidinone. The deprotection and N-acylation is carried out at a temperature between about zero degrees and about room temperature, preferably at about room temperature.
The N-terminus protecting group PG2 in the compounds of structure 13 was removed on treatment with a secondary amine base, preferably piperidine in an inert solvent, preferably N,N-dimethylformamide and then was reacted with phosgene or a phosgene equivalent reagent, to ultimately yield in a two step sequence, the 2,5-dioxoimidazolidines of structure 15. The reaction to give the intermediate isocyanate 14 is conveniently carried out using trichloromethyl chloroformate (diphosgene) in an inert solvent, for example, a halogenated hydrocarbon in the presence of a proton acceptor, for example, pyridine, triethylamine or diisopropylethylamine, preferably diisopropylethylamine at a temperature between about zero degrees and about room temperature, preferably at about room temperature. The thermally induced cyclization of the intermediate isocyanates is accomplished by heating a suspension of the resin bound isocyanates of structure 14 in an inert solvent, for example toluene, at a temperature of from between 50xc2x0 C. and the reflux temperature of the mixture, preferably at about 70xc2x0 C. to give the resin bound compounds of structure 15.
Cleavage of the peptidic residue 15 from the solid support to give the acids of structure 16 is achieved by shaking a suspension of 15 in a strong acid, for example methanesulfonic acid, hydrofluoric acid or trifluoroacetic acid, preferably trifluoroacetic acid optionally in the presence of a cation scavenger and an inert co-solvent, for example dichloromethane. The reaction is conveniently run at a temperature of between about zero degrees and about room temperature, preferably at about room temperature.
Reaction of the acid 16 with R1-NH2 to form the amide of Formula 1 can be carried out under the coupling conditions previously described. The preferred coupling reagent in this instance is HBTU. The reaction is carried out in the presence of a tertiary amine base, such as triethylamine or diisopropylethylamine, preferably diisopropylethylamine in an inert solvent, for example N,N-dimethylformamide or N-methylpyrrolidinone, preferably N-methylpyrrolidinone at from zero degrees to room temperature, most conveniently at room temperature.
Reaction Scheme 3 outlines the preparation of the intermediate N-Fmoc-2-aminothiazole-4-carboxylic acid 3. Initially 9-fluorenylmethoxycarbonyl chloride (18) is reacted with potassium thiocyanate in an inert solvent, preferably ethyl acetate at a temperature of between zero degrees and 5xc2x0 C. Then the reaction is allowed to proceed at a temperature of from zero degrees to 40xc2x0 C., preferably at room temperature to furnish N-Fmoc-thiocyanate (19). Treatment of 19 with a solution of ammonia in an inert solvent, for example methanol or ethanol, preferably methanol at a temperature of from zero degrees to room temperature, preferably zero degrees afforded N-Fmoc-thiourea 20. In the final step, the thiourea 20 is then reacted with bromopyruvic acid to form the thiazole of structure 3. The reaction is conveniently carried out if an inert solvent, such as a cyclic ether, for example tetrahydrofuran or dioxane, preferably dioxane at a temperature of from 40xc2x0 C. to the reflux temperature of the mixture preferably at about 70xc2x0 C.
All of the compounds of Formula I which include the compounds set forth in the Examples, activated glucokinase in vitro by the procedure of Example A. In this manner, they increase the flux of glucose metabolism which causes increased insulin secretion. Therefore, the compounds of Formula I are glucokinase activators useful for increasing insulin secretion.